Stroke kills more than 150,000 people annually and accounts for about one of every 15 U.S. deaths. It is presently the third largest cause of death, ranking behind diseases of the heart and cancer, according to the National Center for Health Statistics.
On average, someone suffers a stroke in the United States every minute; every 3.4 minutes someone dies of a stroke. Based on the Framingham Heart Study, approximately 500,000 people suffer a new or recurrent brain attack each year. Approximately 3,890,000 stroke survivors are alive today. From 1984 to 1994, the death rate from stroke declined 19.8 percent, but the actual number of deaths from brain attack rose slightly.
Stroke is the leading cause of serious, long-term disability in the United States. Stroke accounts for half of all patients hospitalized for acute neurological disease. In 1991-92 one million Americans age 15 and older had disabilities resulting from stroke. According to the Framingham Heart Study, 31 percent of brain attack survivors needed help caring for themselves; 20 percent needed help walking; and 71 percent had an impaired ability to work when examined an average of seven years later. Sixteen percent had to be institutionalized. About 31 percent of people who have an initial stroke die within a year. This percentage is higher among people older than age 65. About two-thirds of men and women who have a brain attack die within 12 years; long-term survivorship is worse in men than in women. 407,000 males and 478,000 females were discharged from hospitals in 1994 after having a stroke.
Stroke is defined as a sudden impairment of body functions caused by a disruption in, e.g., the supply of blood to the brain. For instance, a stroke occurs when a blood vessel bringing oxygen and nutrients to the brain is interrupted by any method including low blood pressure, clogging by atherosclerotic plaque, a blood clot, or some other particle, or when a blood vessel bursts.
Because of the blockage or rupture, part of the brain fails to get the blood flow that it requires. Brain tissue that receives an inadequate supply of blood is said to be ischemic. Deprived of oxygen and nutrients, nerve cells and other cell types within the brain begin to fail, creating an infarct (an area of cell death, or necrosis). As nerve cells (neurons) fail and die, the part of the body controlled by those neurons cannot function either. The devastating effects of ischemia are often permanent because brain tissue has very limited repair capabilities and lost neurons are not usually replaced.
Cerebral ischemia may be incomplete (blood flow is reduced but not entirely cut off), complete (total loss of tissue perfusion), transient or permanent. If ischemia is incomplete and persists for no more than ten to fifteen minutes, neural death might not occur. More prolonged or complete ischemia results in infarction. Depending on the site and extent of the infarction, mild to severe neurological disability or death will follow. Thus, the chain of causality leading to neurological deficit in stroke has two principal components: loss of blood supply, and cell damage and death.
Thrombosis is the blockage of an artery by a large deposit that usually results from the combination of atherosclerosis and blood clotting. Thrombotic stroke (also called cerebral thrombosis) results when a deposit in a brain or neck artery reaches occlusive proportions. Most strokes are of this type.
Embolism is the blockage of an artery or vein by an embolus. Emboli are often small pieces of blood clot that break off from larger clots. Embolic stroke (also called cerebral embolism) occurs when an embolus is carried in the bloodstream to a brain or neck artery. If the embolus reaches an artery that is too small for it to pass through, it plugs the artery and cuts off the blood supply to downstream tissues. Embolic stroke is the clinical expression of this event.
To a modest extent, the brain is protected against cerebral ischemia by compensatory mechanisms that include: collateral circulation (overlapping local blood supplies), and arteriolar auto-regulation (local smooth muscle control of blood flow in the smallest arterial channels). If compensatory mechanisms operate efficiently, slightly diminished cerebral blood flow produces neither tissue ischemia nor abnormal signs and symptoms. Usually, such mechanisms must act within minutes to restore blood flow if permanent infarction damage is to be avoided or reduced. Arteriolar auto-regulation works by shunting blood from noncritical regions to infarct zones.
Even in the face of systemic hypotension, auto-regulation may be sufficient to adjust the circulation and thereby preserve the vitality and function of brain tissue. Alternatively, ischemia may be sufficiently prolonged and compensatory mechanisms sufficiently inadequate that a catastrophic stroke results. With these as the extremes, the gradation of ischemic stroke are described below.
A transient ischemic attack (TIA) is conventionally described as a loss of neurologic function caused by ischemia, abrupt in onset, persisting for less than 24 hours, and clearing without residual signs. Most TIAs last only a few minutes. However, neurologic disability may persist for more than 24 hours before clearing. Such an event is called a reversible ischemic neurological disability (RIND).
An ischemic event that is sufficiently severe to cause persistent disability but that is short of a calamitous stroke, is called a partial nonprogressing stroke (PNS). The penultimate ischemic event, a completed stroke, produces major functional loss. The ultimate ischemic insult is death.
Focal cerebral ischemia must be distinguished from global cerebral hypoxia. In cerebral hypoxia the oxygen supply to the brain is diminished even though blood flow and blood pressure may be normal. Discriminating between diagnoses of patients with acute neurological deficit is critical because patient management takes disparate paths.
There are generally distinct clinical outcomes in stroke versus cerebral hypoxia, although both sets of patients may suffer death or permanent damage. Hypoxia patients who survive past an acute life-threatening period usually show few immediate symptoms of long term damage. Instead, clinical manifestations such as mental deterioration, urinary and fecal incontinence, gait and speech disturbances, tremor and weakness are delayed for periods that may vary from days to weeks. However, as in stroke, progressive loss of neurons due to oxygen deprivation is believed to be a factor in such detrimental effects of hypoxia.
It is an objective of the present application to provide new drugs for treatment and prophylaxis of cerebral ischemia, such as stroke.
It is also an objective of the present application to provide new drugs for treatment and prophylaxis of cerebral hypoxia.
One aspect of the present application relates to a method for limiting damage to neuronal cells by ischemic or hypoxic conditions, e.g., such as may be manifest by a reduction in brain infarct volume, by administering to an individual a hedgehog therapeutic or ptc therapeutic in an amount effective for reducing cerebral infarct volume relative to the absence of administeration of the hedgehog therapeutic or ptc therapeutic.
In other embodiments, the subject method can be used for protecting cerebral tissue of a mammal against the repercussions of ischemia; for treating cerebral infarctions; for treating cerebral ischemia; for treatment of stroke; and/or for treating transient ischemia attacks. In embodiments wherein the patient is treated with a ptc therapeutic, such therapeutics are preferably small organic molecules which mimic hedgehog effects on patched-mediated signals.
Wherein the subject method is carried out using a hedgehog therapeutic, the hedgehog therapeutic preferably a polypeptide including a hedgehog portion comprising at least a bioactive extracellular portion of a hedgehog protein, e.g., the hedgehog portion includes at least 50, 100 or 150 amino acid residues of an N-terminal half of a hedgehog protein. In preferred embodiments, the hedgehog portion includes at least a portion of the hedgehog protein corresponding to a 19 kd fragment of the extracellular domain of a hedgehog protein.
In preferred embodiments, the hedgehog portion has an amino acid sequence at least 60, 75, 85, or 95 percent identical with a hedgehog protein of any of SEQ ID Nos. 10-18, though sequences identical to those sequence listing entries are also contemplated as useful in the present method. The hedgehog portion can be encoded by a nucleic acid which hybridizes under stringent conditions to a nucleic acid sequence of any of SEQ ID Nos. 1-9, e.g., the hedgehog portion can be encoded by a vertebrate hedgehog gene, especially a human hedgehog gene.
In other embodiments, the subject method can be carried out by administering a gene activation construct, wherein the gene activation construct is designed to recombine with a genomic hedgehog gene of the patient to provide a heterologous transcriptional regulatory sequence operatively linked to a coding sequence of the hedgehog gene.
In still other embodiments, the subject method can be practiced with the administration of a gene therapy construct encoding a hedgehog polypeptide. For instance, the gene therapy construct can be provided in a composition selected from a group consisting of a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent.
Where the subject method is carried out using a ptc therapeutic, the therapeutic can be, e.g., a molecule which binds to patched and mimics hedgehog-mediated patched signal transduction. For instance, the binding of the therapeutic to patched may result in upregulation of patched and/or gli expression.
In other embodiments, the ptc therapeutic mimics hedgehog-mediated patched signal transduction by altering the localization, protein-protein binding and/or enzymatic activity of an intracellular protein involved in a patched signal pathway.
In a preferred embodiment, the ptc therapeutic is a small organic molecule, e.g., less than 5 kd, more preferably less than 2.5 kd. For instance, the present invention contemplates the use of small organic molecules which interact with neuronal cells to mimic hedgehog-mediated patched signal transduction.
In a preferred embodiment, the ptc therapeutic is a PKA inhibitor. A variety of PKA inhibitors are known in the art, including both peptidyl and organic compounds. For instance, the ptc therapeutic can be a 5-isoquinolinesulfonamide, such as represented in the general formula: 
wherein,
R1 and R2 each can independently represent hydrogen, and as valence and stability permit a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, xe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94O-lower alkyl, xe2x80x94(CH2)mxe2x80x94O-lower alkenyl; xe2x80x94(CH2)nxe2x80x94Oxe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94SH, xe2x80x94(CH2)mxe2x80x94S-lower alkyl, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)nxe2x80x94Sxe2x80x94(CH2)mxe2x80x94R8, or
R1 and R2 taken together with N form a heterocycle (substituted or unsubstituted);
R3 is absent or represents one or more substitutions to the isoquinoline ring such as a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an amino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido, xe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94OH, xe2x80x94(CH2)mxe2x80x94O-lower alkyl, xe2x80x94(CH2)mxe2x80x94O-lower alkenyl, xe2x80x94(CH2)nxe2x80x94Oxe2x80x94(CH2)mxe2x80x94R8, xe2x80x94(CH2)mxe2x80x94SH, xe2x80x94(CH2)mxe2x80x94S-lower alkyl, xe2x80x94(CH2)mxe2x80x94S-lower alkenyl, xe2x80x94(CH2)nxe2x80x94Sxe2x80x94(CH2)mxe2x80x94R8;
R8 represents a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and
n and m are independently for each occurrence zero or an integer in the range of 1 to 6.
Exemplary PKA inhibitors of this class include N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline-sulfonamide and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine. Other PKA inhibitors which can be used in the subject method include KT5720; and PKA Heat Stable Inhibitor (isoform xcex1).
In yet other embodiments of the present invention, the ptc therapeutic alters the level of expression of a hedgehog protein, a patched protein or another protein involved in the intracellular signal transduction pathway of patched. In this regard, the ptc therapeutic can be an antisense construct which inhibits the expression of a protein which is involved in the signal transduction pathway of patched and the expression of which antagonizes hedgehog-mediated signals. For example, the antisense molecule can be one which hyridizes to a patched transcript or genomic sequence, such as 5xe2x80x2-GTCCTGGCGCCGCCGCCGCCGTCGCC, 5xe2x80x2-TTCCGATGACCGGCCTTTCGCGGTGA or 5xe2x80x2-GTGCACGGAAAGGTGCAGGCCACACT (SEQ ID NOS: 24-26).
In yet other embodiments, the subject method can be carried out with a gene activation construct, which construct recombines with a genomic hedgehog gene of the patient, e.g., to form a chimeric gene, providing a heterologous transcriptional regulatory sequence operatively linked to a coding sequence of the hedgehog gene. The transcriptional regulatory sequence can provide for constitutive or inducible expression of the hedgehog gene.
The subject method can be used as part of a treatment for stroke, e.g., thrombotic stroke and/or embolic stroke.
The subject method can also be used to treat hypoxic conditions which otherwise result in cerebral hypoxia.
The subject method can be used prophylactically or as an ipso facto treatment. It can be used to treat patients who are hypotensive.
The subject method can also be used as part of a therapy including administering one or more of an anticoagulant, an antiplatelet agent, a thrombin inhibitor, and/or a thrombolytic agent, and/or in conjunction with vascular surgery, e.g., carotid endarterectomy.
In preferred embodiments, the subject method results in at least a 25%, 50%, 70%, 75%, or 90% reduction in cerebral infarct volumes relative to the absence of treatment with the therapeutic, e.g., as measured in a stroke model such as the MCAO model.